Biocatalytic and Chemo-Enzymatic Synthesis of Quinolines and 2-Quinolones by Monoamine Oxidase (MAO-N) and Horseradish Peroxidase (HRP) Biocatalysts

The oxidative aromatization of aliphatic N-heterocycles is a fundamental organic transformation for the preparation of a diverse array of heteroaromatic compounds. Despite many attempts to improve the efficiency and practicality of this transformation, most synthetic methodologies still require toxic and expensive reagents as well as harsh conditions. Herein, we describe two enzymatic strategies for the oxidation of 1,2,3,4-tetrahydroquinolines (THQs) and N-cyclopropyl-N-alkylanilines into quinolines and 2-quinolones, respectively. Whole cells and purified monoamine oxidase (MAO-N) enzymes were used to effectively catalyze the biotransformation of THQs into the corresponding aromatic quinoline derivatives, while N-cyclopropyl-N-alkylanilines were converted into 2-quinolone compounds through a horseradish peroxidase (HRP)-catalyzed annulation/aromatization reaction followed by Fe-mediated oxidation.


General Information
Unless noted, all solvents and commercially available reagents were purchased from Sigma Aldrich and used as without further purifications. Horseradish peroxidase (HRP) was purchased from Alfa Aesar (240 units/mg dry weight). 1,2,3,4-Tetrahydroquinolines were purchased from Fluorochem. 1 H and 13 C Nuclear Magnetic Resonance (NMR) spectra were recorded using a Bruker Ascend 400 spectrometer at 298 K. Chemical shifts (δ) are reported in ppm, referenced to tetramethylsilane. Coupling constants (J) are reported in Hertz. Splitting patterns are abbreviated as follows: singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m). TLC was performed using commercially available pre-coated plated and visualized with UV light at 254 nm. Flash column chromatography was carried out using Sigma Aldrich silica gel particle size, 40-63 µm particle size 60 Å. Biocatalytic reactions (MAO) were performed by shaking the mixtures contained in 15 mL Falcon tubes using a Grant Bio™ PSU-10i Orbital Platform Shaker. A JouanB4 centrifuge with exchangeable buckets was used to centrifuge and isolate the biocatalytic products. HRMS (high-resolution mass) were measured on a Thermo Q-Exactive mass spectrometer with an EI/ESI/APCI source. The NMR data of the known compounds were consistent with the data already reported in literature.  Step 1: A heavy wall cylindrical vessels (15 mL, Synthware) equipped with a Teflon coated magnetic stir bar was loaded with Pd(dba) 2 (20 mg), BrettPhos (128 mg) and t BuOK (1.3 g). The vessel was purged with N 2 (three times), and then toluene (5 mL), bromobenzene (3 mmol) and cyclopropylamine (6 mmol) were added. The vessel was purged with N 2 for 1 minute and screw-capped. The reaction mixture was heated at 80 o C (pre-heated oil bath) for 24 hours. Then the reaction mixture was cooled to room temperature, diluted with ethyl acetate and filtered. The filtrate was concentrated in vacuo and used for the next step without any further purification.
Step 2: To a solution of the obtained N-cyclopropylbenzenamine in 25 mL acetone was added K 2 CO 3 (12 mmol) and MeI (18 mmol). The mixture was then stirred at room temperature for 1-3 days. Once the reaction was completed (by TLC monitoring) the solvent was removed in vacuo and the residue was purified by flash column chromatography (hexane/EtOAc = 80/1) to afford the desired products 3.

Preparation of the whole cell MAO-N biocatalysts
MAO-N biocatalysts (monoamine oxidase from Aspergillus niger) were expressed in E. coli cells according to previously reported procedures. MAO-N D5 was produced in E. coli BL21(DE3) MAO-N D9 was produced in E. coli BL21(DE3) MAO-N D11 was produced in E. coli C43(DE3) In all cases, an overnight 10 mL starter culture of each clone was grown in LB broth + Ap (100 μg/ml) at 37 o C, 200 rpm. The starter culture was then inoculated into 1 L of Auto Induction Media Super Broth Base including trace elements (Formedium Ltd, UK) + Ap (100 μg/ml), in a 2 L baffled flask, and grown at 30 o C, 180 rpm for 2 days. Cells were then harvested by centrifugation at 4000 x g for 10 min at 4 o C. The supernatant was discarded and the cell pellet was resuspended in 10 mL of 18.2 MΩ/cm H 2 O. The resuspended cells were then frozen and freeze-dried. Typically 4 g of lyophilized E. coli cells were obtained from a 600 mL culture. In a Falcon tube (15 mL), freeze-dried whole cells of E.coli expressing recombinant monoamine oxidase MAO-D11 (190 mg obtained from bulk production) or pure MAO-N D11 were suspended in buffer (Na 2 HPO 4 /NaH 2 PO 4 , pH = 7.8, 1.0 M) (3.0 mL). Thereafter, the appropriate 1,2,3,4-tetrahydroquinoline substrates (0.2 mmol) dissolved in DMSO (50 μL) was added. After addition, the mixture was incubated at 37 ºC and shaken at 160 rpm for 7 days. Then, the reaction mixture was extracted with ethyl acetate (3 x 3.0 mL) and centrifuged at 4000 x g for 10 minutes. The organic layers were then separated and combined and then dried over anhydrous MgSO 4 . After filtration and removal of the solvents in vacuo, the crude product was analyzed through 1 H-NMR and the conversion value was determined by integration. Taking 2b and 2e (1 mmol scale) as example, the crude products were finally purified by column chromatography (hexane/EtOAC = 50/1) to afford the pure quinoline products.  In a glass rolled rim vial (5 mL) equipped with a magnetic stirring bar, N-cyclopropyl-N-methylaniline 3 was suspended in buffer (Na 2 HPO 4 /NaH 2 PO 4 , pH = 5.5, 0.4 M) (2.0 mL). Thereafter, 20 μL acetone, 100 μL HRP solution (4 mg per 1 mL buffer, 240 U/mg) and 20 μL H 2 O 2 (30%) were added successively, while stirring. The reaction mixture was stirred at room temperature for 0.5 h and then additional 50μL HRP solution was added. The reaction was detected by TLC and the crude product was analyzed through 1 H-NMR and the yield was calculated by integration with sodium 4-methylbenzenesulfonate as internal standard. Step 1: Following the previous procedure, the scale-up of the reaction to 0.32 mmol was carried out accordingly. However, solid HRP (2.4 mg) was added directly to the reaction mixture.
Step 2: After the full consumption the starting materials 3 detected by TLC, K 3 Fe(CN) 6 (10 equiv.) and NaOH (34 equiv.) in 2 mL water were added to the reaction mixture. The obtained suspension was stirred at room temperature overnight. The reaction mixture was extracted with EtOAc (3 x 20 mL) and the combined organic layer was washed with saturated salt solution and water. Then the organic layer was dried over anhydrous MgSO 4 . After filtration and removal of the solvents in vacuo, the crude product was purified by column chromatography (hexance/EtOAc = 40/1).

In Situ EPR Experiments
The X-band continuous-wave EPR spectra were recorded on a Magnettech ESR5000 spectrometer (Bruker) equipped with a TCH04 temperature controller and a quartz Dewar insert. For each experiment, the microwave power was set to 10 mW and a field modulation of 1 mT at 100 kHz was used; 4 traces recorded with a field sweep rate of 1.67 mT/s were accumulated. The EPR signals clearly indicate the involvement of a radical in this process, which disappeared without 3a. Based on these results, this EPR signals presumably should be the corresponding radical from HRP. However, the EPR signals were unable to be precisely identified due to their insufficient intensity. Figure S3. ESR spectrum of the HRP biocatalyzed cyclization/aromatization of 3a

Computation Methods
All molecular docking studies were performed on a Viglen Genie Intel®CoreTM i7-3770 vPro CPU@ 3.40 GHz x 8 running Ubuntu 18.04. Molecular Operating Environment (MOE) 2022.10, Maestro (Schrödinger Release 2020-2) and Multiwfn were used as molecular modelling software. 1,2,3 The MAO-N D11 structure was downloaded from the PDB data bank (http://www.rcsb.org/; PDB code 3ZDN). The protein was preprocessed using the Schrödinger Protein Preparation Wizard by assigning bond orders, adding hydrogens and performing a restrained energy minimisation of the added hydrogens using the OPLS_2005 force field. Ligand structures were built with MOE and then prepared using the Maestro LigPrep tool by energy minimising the structures (OPLS_2005 force filed), generating possible ionization states at pH 7±2, generating tautomers and low-energy ring conformers. A 11 Å docking grid (inner-box 10 Å and outer-box 21 Å) was prepared using as centroid the co-crystallized FAD. Molecular docking studies were performed using Glide SP precision keeping the default parameters and setting 10 as number of output poses per input ligand to include in the solution. The output poses were saved as mol2 file. The docking results were visually inspected for their ability to bind the active site in MOE. Quantum-chemical computations were performed using Gaussian. 3 Molecular structures were optimised at the B3LYP/6-31G(d) level of theory, in the gas phase, while the ESP was calculated using a constant electronic density of 0.002 au. The ESP values were shown by means of coloured maps on isodensity surface (range between -4.0e -2 and 4.0e -2 a.u). The nucleophilicity index N prediction was performed using Multiwfn based on the HOMO energies is obtained within the Kohn-Sham scheme, and defined as N = E HOMO(Nu) - E HOMO(TCE) 5 .

Characterization data for the new compounds
References for known compounds. The data of the known quinoline and quinolinium compounds prepared in this work were compared and found in agreement with those reported in literature. The references for the known compounds are reported in the Table S1. Table S1. References for known compounds.